Amperometric Immunoassay Research Articles

Wire-in-Tube IrOx Architectures: Alternative Label-Free Immunosensor for Amperometric Immunoassay toward α-Fetoprotein.

A sensitive, label-free immunosensor based on iridium oxide (IrOx, 0≤x≤2) nanofibers, which were synthesized through a simple one-spinneret electrospinning method, was first developed for immunoassay of the cancer biomarker α-fetoprotein (AFP). The specific wire-in-tube nanostructure could be obtained and the composition of IrOx nanofibers also could be controlled through changing the annealing temperature. The unique structure and properties of IrOx nanofibers obtained at 500 °C not only led to increased electrode surface area and accelerated electron transfer kinetics but also could provide a highly stable matrix for the convenient conjugation of biomolecules together with chitosan (CS). The good electrochemical properties of the IrOx-nanofiber-modified immunosensor allowed one to detect AFP over a wide concentration range from 0.05 to 150 ng/mL, with a detection limit of 20 pg/mL. The proposed immunosensor also has been used to determine AFP in human serum with satisfactory results. The present protocol was shown to be quite promising for clinical screening of cancer biomarkers and point-of-care diagnostics applications.

Amperometric magnetoimmunoassay for the determination of lipoprotein(a)

A highly sensitive amperometric magnetoimmunoassay for rapid determination of lipoprotein(a) (Lp(a)), an important predictor of cardiovascular disease risk, in human serum, is described. It uses a sandwich configuration involving selective capture antibody [antiLp(a)] and biotinylated detector antibody [biotin-antiLp(a)], and a streptavidin-HRP conjugate on carboxy-modified magnetic beads (HOOC-MBs). The resulting MBs bearing the sandwiched immunoconjugates were captured by a magnet placed under the working electrode surface of a disposable screen-printed carbon electrode and the extent of the affinity reaction was monitored amperometrically at −0.20 V (vs a silver pseudo-reference electrode) in the presence of hydroquinone as an electron transfer mediator and upon addition of H2O2 as the enzyme substrate. The method exhibited a wide linear response range (from 0.01 to 0.5 μg mL−1), a detection limit of 4 ng mL−1, and an excellent selectivity over other serum components. The utility of the immunoassay was demonstrated by analyzing a reference serum containing a certified quantity of Lp(a). The performance of this magnetoimmunoassay compares favorably to that of an integrated amperometric immunoassay described earlier.

Amperometric magnetoimmunoassay for the direct detection of tumor necrosis factor alpha biomarker in human serum

An amperometric immunoassay for the determination of tumor necrosis factor alpha (TNFα) protein biomarker in human serum based on the use of magnetic microbeads (MBs) and disposable screen-printed carbon electrodes (SPCEs) has been developed. The specifically modified microbeads were magnetically captured on the working electrode surface and the amperometric responses were measured at −0.20V (vs. Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as electron transfer mediator and H2O2 as the enzyme substrate. After a thorough optimization of the assay, extremely low limits of detection were achieved: 2.0pgmL−1 (36fM) and 5.8pgmL−1 (105fM) for standard solutions and spiked human serum, respectively. The simplicity, robustness and this clinically interesting LOD proved the developed TNFα immunoassay as a good contender for real clinical application.

Label-free immunosensor based on Pd nanoplates for amperometric immunoassay of alpha-fetoprotein

In this paper, Pd nanoplates were used as a kind of electrode materials for fabrication of an electrochemical immunosensor, which was applied for detection of cancer biomarker alpha-fetoprotein (AFP). Thanks to the unique structure and properties of Pd nanoplates, the antibody of AFP (Ab) was effectively immobilized onto the surface of the Pd nanoplates modified glassy carbon electrode (GCE). Moreover, the good electrochemical properties of Pd nanoplates greatly improved the electronic transmission rate and enhanced the electrochemical signal, which led to an increase of the detection sensitivity. Based on the specific antibody–antigen interaction, a label-free immunosensor based on Pd nanoplates was developed for sensing of AFP. The current method allows us to detect AFP over a wide concentration range from 0.01 to 75.0ng/mL with a detection limit of 4pg/mL. The proposed immunosensor has been used to determine AFP in human serum with satisfactory results.

Label-free electrochemical immunoassay for ultrasensitive detection of norethindrone

A label-free electrochemical immunoassay based on the graphene sheet (GS)–carboxymethyl chitosan (CMC)–thionine (TH) matrix (GS–CMC–TH matrix) for the sensitive determination of norethindrone antigen has been prepared. GS has excellent electrochemical activity and the synergistic effect between GS and TH in the CMC matrix was investigated. The anti-norethindrone was immobilized onto the GS–CMC–TH matrix modified glassy carbon electrode (GC) through covalent binding. The stable thin film formed with the GS–CMC–TH matrix on the surface of GC indicated high electroactivity, and it was found that the electroactivity of TH was greatly improved in the presence of GS due to the synergistic effect between GS and TH. After anti-norethindrone was conjugated onto the film of GS and TH through N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, the modified electrode could be used as an amperometric immunoassay for the detection of norethindrone antigen. The specific antibody-antigen immunocomplex formed on the electrode resulted in the decrease of amperometric signal of the electrode. The amperometric signal decreased linearly with norethindrone antigen concentration in the range of 0.01–4.2 ng/cm3 with a detection limit of 2.01 pg/cm3. The immunoassay had the advantages of high sensitivity, good selectivity and stability, and was applied to the analysis of norethindrone antigen in aquatic product with satisfactory results. .

Amperometric Detection ofBacillus anthracisSpores: A Portable, Low-Cost Approach to the ELISA

Antibody-based detection assays are generally robust, a desirable characteristic for in-the-field use. However, to quantify the colorimetric or fluorescent signal, these assays require expensive and fragile instruments which are ill-suited to in-the-field use. Lateral flow devices (LFDs) circumvent these barriers to portability but suffer from poor sensitivity and subjective interpretation. Here, an antibody-based method for detectingBacillus anthracisspores via amperometric signal generation is compared to ELISA and LFDs. This amperometric immunoassay uses antibody conjugated to magnetic beads and glucose oxidase (GOX) along with the electron mediator 2, 6-dichlorophenolindophenol (DCPIP) for production of a measurable current from a 0.4 V bias voltage. With similar sensitivity to ELISA, the assay can be completed in about 75 minutes while being completely powered and operated from a laptop computer. Immunoassay amperometry holds promise for bringing low-cost, quantitative detection of hazardous agents to the field.

INFLUENCE OF THE FUNGAL MICROFLORA ON THE COURSE OF PERENNIAL ALLERGIC RHINITIS IN CHILDREN

Background. The purpose of our research was to study the influence of fungal microflora for perennial rhinitis in children to improve the treatment of this disease. Methods. 79 children aged from 7 to 18 years with perennial allergic rhinitis (PAR) with fungal colonization of the nasal mucosa were examined. Patients underwent clinical, allergy, culturing mycological examination of the mucous membranes of the nose, as well as determination of circulating Candida antigen in serum by ampero-metric enzyme immunoassay sensor. Results. Fungi Candida were dominated — in 75,9% of cases in the structure of mycotic colonization, while mold fungi Aspergillus were observed rarely (24,1%). Colonization of the nasal mucosa with fungi Candida in children with PAR makes disease severe and symptoms become more painful that manifest severe nasal congestion, abundant mucus. PAR patients with nasal colonization of the fungi genus Candida in 58,3% of cases in the circulating blood serum had Candida antigen, that indicating the development of invasive Candida infections. That's why therapy of this disease must be improved. Inclusion of antimicrobial agents to the treatment with further traditional therapy of PAR leads to an overall therapeutic effect in 77,1% of cases. Conclusion. In patients with PAR fungal microflora aggravates the disease and the treatment needs to be improved with the inclusion of antibiotics, which lead to high clinical improvement.

Amperometric homogeneous competitive immunoassay in a perfluorocarbon emulsion oxygen therapeutic (PEOT)

The effect of a perfluorocarbon emulsion oxygen therapeutic (PEOT) on the detection of the drugs theophylline and phenytoin was explored using a commercial enzyme multiplied immunoassay technique (EMIT®). The EMIT technique is based on the enzymatic production of NADH, which is typically detected in serum samples spectrophotometrically. Here, amperometry using the rotating disk electrode on a single drop of solution is demonstrated to detect theophylline and phenytoin in the presence of PEOT. In the study, 2,6-dichloroindophenol (DCIP) added to the immunoassay mixture is reduced by the NADH to DCIPH2. Oxidation of DCIPH2 is monitored electrochemically at +200 mV using a glassy carbon rotating disk electrode. Slopes of amperograms are proportional to the concentration of drug in the immunoassay sample. This technique yields excellent quantitative data in the therapeutic range for both drugs in 2-20% PEOT.

A Direct Competitive Homogeneous Immunoassay for Progesterone – the Redox Quenching Immunoassay

AbstractA direct competitive amperometric immunoassay format for the detection of haptens and proteins was developed. The method is based on the quenching of electroactivity of ferrocenium, which is coupled to the antigen and used as the primary reporter, upon binding to a monoclonal anti‐ferrocenium antibody, which is coupled to the detection antibody and used as a secondary reporter. A separation‐free progesterone immunoassay with a lower detection limit of 1 ng mL−1 (3.18 nmol L−1) in 1 : 2 diluted blood serum was realised by combining two bifunctional conjugates, a ferrocenium‐PEG‐progesterone tracer and a bioconjugate of one anti‐progesterone and one anti‐ferrocenium antibody. The immune complex is formed within 30 s upon addition of progesterone, resulting in a total analysis time of 1.5 min.

A Label-Free Amperometric Immunoassay for Thrombomodulin Using Graphene/Silver-Silver Oxide Nanoparticles as a Immobilization Matrix

A simple, sensitive, and label-free electrochemical immunosensor has been developed for the measurement of serum thrombomodulin (TM), an endothelial glycoprotein which is associated with the progression and metastasis of tumors. At first, the graphene nanosheets, which were dispersed in Nafion solution, were used to coat the bare gold electrode. Then, silver-silver oxide nanoparticles (Ag-Ag2O NPs) were immobilized on the graphene-modified electrode by a one-step electrochemical deposition method. Lastly, a thrombomodulin antibody (anti-TM) was attached via amido-Ag affinity. This strategy combines graphene/Ag-Ag2O NPs hybrid materials as an immobilization matrix and Ag-Ag2O NPs also as an electrochemical signal indication reagent. The main advantage of this strategy has two important aspects. One is the high stability and unique electronic properties of the graphene nanostructure. The other is the use of Ag-Ag2O NPs as the immobilization matrix and redox probes, thus avoiding the laborious labeling protein operation. Using this strategy, the concentration of TM in the range of 0.1 to 20 ng/mL was detected, with a detection limit of 31.5 pg/mL (at 3σ). The proposed methodology demonstrates that the nanocomposite film composed of graphene and Ag-Ag2O NPs is a potential for biosensor applications.

An Amperometric Immunoassay for Human Chorionic Gonadotrophin Based on DNA-Multiwalled Carbon Nanotubes and Methylene Blue as Sensing Interface

An Amperometric Immunoassay for Human Chorionic Gonadotrophin Based on DNA-Multiwalled Carbon Nanotubes and Methylene Blue as Sensing Interface

Field-effect amperometric immuno-detection of protein biomarker

The field-effect enzymatic detection technique has been applied to the amperometric immunoassay of the cancer biomarker, carcinoma antigen 125 (CA 125). The detection adopted a reagentless approach, in which the analyte, CA 125, was immobilized on the detecting electrode, which was modified using carbon nanotubes, and the detection signal was obtained by measuring the reduction peak current of the enzyme that was used to label the antibody. A gating voltage was applied to the detecting electrode, inducing increase in the signal current and therefore providing amplification of the detection signal. The voltage-controlled signal amplification of the detection system has increased the sensitivity and lowered the detection limit of the system. A detection limit of 0.9U/ml was obtained in the work.

Determination of Pharmaceuticals Based on Indole Alkaloids with Amperometric DNA-Sensors and Enzyme Immunoassay Test-System

Bioaffine methods were developed for determining indole-containing alkaloids ajmaline and vincristine. These methods are based on the proposed amperometric DNA-sensors and on immunoenzyme test-system with a spectrophotometric indication of the analytical signal. The complexing between ajmaline and immobilized native DNA (n-IDNA) allows effective preliminary concentration from test solutions on the biosensor. The time of analysis is 25–30 min, limit of detection (LOD) for ajmaline is 1.0 × 10−10 M. The test-system utilized the immunological reaction of ajmaline with its antibodies and the enzyme marker, horseradish peroxidase, LOD is 1.5 × 10−9 M. Anti-cancer vincristine interaction with immobilized renatured DNA (r-IDNA) was studied and determination was carried out using an amperometric DNA-sensor. Bioaffine membrane drug concentration and reactivation of the sensor was performed. The affinity binding constant Kbind for vincristine-r-IDNA complex calculated by Scatchard's method was found to be high enough [(5.0 ± 0.4) × 105 l/mol] confirming high specificity of the complexing with r-IDNA. The duration of the assay is 40 min. The developed method is characterized with an LOD of 1.1 × 10−9 M, with the lack of the need for long sample processing. The pharmaceuticals were determined by those methods in model solutions of blood serum and in tablets and solutions for injections.

Gold–silver–graphene hybrid nanosheets-based sensors for sensitive amperometric immunoassay of alpha-fetoprotein using nanogold-enclosed titania nanoparticles as labels

A new sandwich-type electrochemical immunosensor with enhanced sensitivity was developed for detection of alpha-fetoprotein (AFP, as a model analyte) in biological fluids by using nanogold-enclosed titania nanoparticle (AuTi)-labeled secondary antibody on a gold–silver–graphene hybrid nanosheet (AuAgGP)-functionalized glassy carbon electrode (GCE). The presence of the AuAgGP nanosheets not only enhanced the immobilized amount of biomolecules, but also improved the electrochemical properties of the immunosensor. With the aid of AuTi nanolabels, the electrochemical signal was greatly amplified in comparison with pure nanogold or titania-based labels. Under optimal conditions, the sensitivity and dynamic range of the immunosensor were evaluated by using the labeled horseradish peroxidase on the AuTi as trace and H 2O 2 as enzyme substrate, and exhibited a wide dynamic range of 0.001–200 ng mL −1 with a low detection limit (LOD) of 0.5 pg mL −1 AFP (at 3 σ). Both the intra- and inter-assay coefficients of variation were less than 10%. The current of the immunosensor at 13th day was as much as 90% of the initial current. In addition, the methodology was evaluated for 8 positive serum specimens obtained from hepatocarcinoma patients and 19 negative sera, and validated with the commercially available Roche 2010 Electrochemiluminescent (ECL) Automatic Analyzer. No significant differences at the 95% confidence level were encountered between two methods.

Carbon nanotube-based symbiotic coaxial nanocables with nanosilica and nanogold particles as labels for electrochemical immunoassay of carcinoembryonic antigen in biological fluids

A feasible and practicable amperometric immunoassay strategy for sensitive screening of carcinoembryonic antigen (CEA) in human serum was developed using carbon nanotube (CNT)-based symbiotic coaxial nanocables as labels. To construct such a nanocable, a thin layer of silica nanoparticles was coated on the CNT surface by sonication and sol–gel methods, and then colloidal gold nanoparticles were assembled on the amino-functionalized SiO2/CNTs, which were used for the label of horseradish peroxidase-anti-CEA conjugates (HRP-anti-CEA-Au/SiO2/CNT). In the presence of analyte CEA, the sandwich-type immunocomplex was formed on an anti-CEA/Au/thionine/Nafion-modified glassy carbon electrode by using HRP-anti-CEA-Au/SiO2/CNTs as detection antibodies. To embody the advantages of the protocol, the analytical properties of variously modified electrodes were compared in detail on the basis of different nanolabels. Under optimal conditions, the cathodic peak currents of the electrochemical immunosensor were proportional to the logarithm of CEA concentration over the range from 0.01 to 12ngmL−1 in pH 5.5 HAc-NaAc containing 5mM H2O2. At a signal-to-noise ratio of 3, the detection limit (LOD) is 5pgmL−1 CEA. Intra- and inter-assay coefficients of variation were below 9.5%. Meanwhile, the selectivity and stability of the immunosensor were acceptable. In addition, the technique was evaluated by spiking CEA standards in pH 7.4 PBS and with 35 clinical serum specimens, receiving excellent accordance with results from commercially available electrochemiluminescent enzyme-linked immunoassay.

A graphene-based Au(111) platform for electrochemical biosensing based catalytic recycling of products on gold nanoflowers

A novel, enzyme-free amperometric immunoassay of biomarkers with sensitive enhancement was designed by using gold nanoflower-labeled detection antibodies toward the catalytic reduction of p-nitrophenol and redox cycling of p-aminophenol on a graphene-based Au(111) platform.

Development of amperometric immunoassays (AIAs) for calpastatin and μ-calpain with possible applications in the biomedical field

Due to the involvement of calpains and calpastatin in the pathology of several diseases, it is important to create an analytical tool for their accurate and sensitive measurements. In this work, an amperometric immunoassay (AIA) was developed and optimized for quantification of two key proteins from the calpain proteolytic system. The detection is based on the measurement of a steady-state current on screen printed electrodes due to the reduction of the enzymatic product resulting from specific immunoassays, where the measured current is proportional to the concentration of the target analyte. The optimized assays with dynamic ranges from 0.09 to 6ng/mL for calpastatin and 0.04 to 5μg/mL for calpain, offer simplicity, sensitivity and possibility for miniaturization.

Electrochemical immunosensor detection of urinary lactoferrin in clinical samples for urinary tract infection diagnosis

Urine is the most abundant and easily accessible of all body fluids and provides an ideal route for non-invasive diagnosis of human diseases, particularly of the urinary tract. Electrochemical biosensors are well suited for urinary diagnostics due to their excellent sensitivity, low-cost, and ability to detect a wide variety of target molecules including nucleic acids and protein biomarkers. We report the development of an electrochemical immunosensor for direct detection of the urinary tract infection (UTI) biomarker lactoferrin from infected clinical samples. An electrochemical biosensor array with alkanethiolate self-assembled monolayer (SAM) was used. Electrochemical impedance spectroscopy was used to characterize the mixed SAM, consisted of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol. A sandwich amperometric immunoassay was developed for detection of lactoferrin from urine, with a detection limit of 145 pg/ml. We validated lactoferrin as a biomarker of pyuria (presence of white blood cells in urine), an important hallmark of UTI, in 111 patient-derived urine samples. Finally, we demonstrated multiplex detection of urinary pathogens and lactoferrin through simultaneous detection of bacterial nucleic acid (16S rRNA) and host immune response protein (lactoferrin) on a single sensor array. Our results represent first integrated sensor platform capable of quantitative pathogen identification and measurement of host immune response, potentially providing clinical diagnosis that is not only more expeditious but also more informative than the current standard.

Novel polymeric bionanocomposites with catalytic Pt nanoparticles label immobilized for high performance amperometric immunoassay

We report on the aqueous preparation of novel polymeric bionanocomposites (PBNCs) with polydopamine (PDA) as an efficient matrix to support antibody and uniformly dispersed abundant Pt nanoparticles (PtNPs) for high performance sandwich-type amperometric immunoassay. We prepare three kinds of PBNCs (PBNCs 1, 2, and 3) via chemical polymerization synthesis (1) and further adsorption (2) or glutaldehyde (GA) covalent crosslinking (3) of antibody. Transmission electron microscopy, atomic force microscopy, and quartz crystal microbalance are used to characterize the PBNCs. The results show that PDA is an excellent matrix to support antibody and PtNPs due to its high biocompatibility, adsorbability, and processibility, and the thus-prepared PBNCs are of high immuno-recognition efficiency and high catalytic activity toward H2O2 reduction. Then, we examine the applicability of the prepared PBNCs for sandwich-type amperometric immunosensing using a model immuno-pair of human immunoglobulin G (hIgG) and anti-hIgG, and obtain the signal by detecting the electro-reduction of H2O2 catalyzed by the PtNPs label. The constructed immunosensors using PBNCs 1, 2 and 3 exhibit detection limits of 0.068, 0.037, and 0.018ngmL−1, respectively, being competitive with or better than the reported analogues. Our immunosensors also present good reproducibility, stability, regeneration ability, specificity, and satisfactory feasibility for target assay in clinical human serum samples. The suggested protocol for the preparation of PBNCs with high signal-transduction ability is expected to find wide bioassay applications.

Ionic liquids-doped organic-inorganic hybrid film for electrochemical immunoassay for hepatitis B surface antigen

A new mediator-free amperometric immunoassay for hepatitis B surface antigen (HBsAg) in human serum was designed by means of immobilizing horseradish peroxidase-hepatitis B surface antibodies conjugates (HRP-HBsAb) on ionic liquids-doped organic-inorganic hybrid film. The composite film including magnetic nanogold particles and nanoalumina particles provides a friendly microenvironment for the immobilization of biomolecules. The presence of ionic liquids enhances the electron communication between the immobilized biomolecules and the base electrode and improves the sensitivity of the electrochemical immunoassay. With a non-competitive immunoassay format, the formation of the immunocomplex between the immobilized HRP-HBsAb and HBsAg in sample solution exhibited a barrier of direct electrical communication between the immobilized HRP and the base electrode and changed the bioelectrocatalytic properties of the immobilized HRP towards H2O2 in the detection solution. Under optimal conditions, the developed immunosensors displayed a good current response in a dynamic range from 1.2 to 430 ng/mL with a limit of detection of 0.3 ng/mL HBsAg (at 3s). The proposed immunosensors have good precision, high sensitivity, acceptable stability, and reproducibility and could be used for the HBsAg detection in human serum with consistent results in comparison with those obtained by the enzyme-linked immunosorbent assay method.